Suppressing posttranslational gluconoylation of heterologous proteins by metabolic engineering of Escherichia coli

Abstract

Minimization of chemical modifications during the production of proteins for pharmaceutical and medical applications is of fundamental and practical importance. The gluconoylation of heterologously expressed protein which is observed in Escherichia coli BL21(DE3) constitutes one such undesired posttranslational modification. We postulated that formation of gluconoylated/phosphogluconoylated products of heterologous proteins is caused by the accumulation of 6-phosphogluconolactone due to the absence of phosphogluconolactonase (PGL) in the pentose phosphate pathway. The results obtained demonstrate that overexpression of a heterologous PGL in BL21(DE3) suppresses the formation of the gluconoylated adducts in the therapeutic proteins studied. When this E. coli strain was grown in high-cell-density fed-batch cultures with an extra copy of the pgl gene, we found that the biomass yield and specific productivity of a heterologous 18-kDa protein increased simultaneously by 50 and 60%, respectively. The higher level of PGL expression allowed E. coli strain BL21(DE3) to satisfy the extra demand for precursors, as well as the energy requirements, in order to replicate plasmid DNA and express heterologous genes, as metabolic flux analysis showed by the higher precursor and NADPH fluxes through the oxidative branch of the pentose phosphate shunt. This work shows that E. coli strain BL21(DE3) can be used as a host to produce three different proteins, a heterodimer of liver X receptors, elongin C, and an 18-kDa protein. This is the first report describing a novel and general strategy for suppressing this nonenzymatic modification by metabolic pathway engineering.

FIG. 1.

In vitro activities of the PGL enzyme from P. aeruginosa and E. coli batch cultures. Broth samples were collected at mid-exponential phase and processed to measure the in vitro PGL activities exhibited by P. aeruginosa and E. coli BL21(DE3). The enzyme activity is expressed in units (mmol NADPH/liter) per minute per gram (dry weight). CDW, cell dry weight.

FIG. 2.

Evidence of adduct formation during production of LXR/RXR, elongin C, and the 18-kDa product. Broth samples were collected at the end of the fermentation and processed as described in Materials and Methods. The gluconoylated and phosphogluconoylated LXR derivatives (A) and the gluconoylated elongin C derivative (B) were evaluated by LC/MS analysis. In the case of the 18-kDa protein (C) the gluconoylated adduct was detected in whole-cell lysates using a high-resolution RP-HPLC method. The circled peaks are product derivatives exhibiting different extents of gluconoylation.

FIG. 3.

Evidence of adduct removal during production of LXR/RXR, elongin C, and the 18-kDa product. Cultures of the recombinant E. coli BL21(DE3)+PGL strain for production of LXR/RXR, elongin C, and 18-kDa protein were grown in parallel with corresponding “control” fermentations of the BL21(DE3) strain without PGL overexpression. The only difference between runs for product synthesis was the presence of the low-copy-number pECO-1pgl plasmid. As noted above, each comparative study included an assessment of the adduct levels by either LC/MS or high-resolution RP-HPLC analyses.